CN103290054A - Recombinant factor C from Tachypleus tridentatus expressed by insect cells - Google Patents
Recombinant factor C from Tachypleus tridentatus expressed by insect cells Download PDFInfo
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Abstract
The invention relates to the field of bioengineering, in particular to a method for recombining and expressing a gene of a factor C from Tachypleus tridentatus in insect cells so as to detect and remove endotoxin. The method comprises the steps of preparing a recombinant rhabdovirus of the gene of the factor C from Tachypleus tridentatus, transfecting the insect cells, detecting the expression of the recombinant factor C from Tachypleus tridentatus in the insect cells, and the like. The recombinant factor C from Tachypleus tridentatus, prepared by the method, provides a precondition for researching the binding activity of endotoxin of the factor C, and also lays a foundation for the application of the factor C in detection and removal of the endotoxin.
Description
Technical field:
The present invention relates to bioengineering field, preparation specifically contains the recombinant baculovirus of Tachypleus tridentatus C factor gene, and is used for transfection insect cell, the Tachypleus tridentatus C factor of express recombinant.
Background technology:
The clinical transfusion reaction is maximum with pyrogen reaction harm, and incidence is the highest.Intracellular toxin (is the lipopolysaccharide molecule of gram-negative bacteria cell wall, lipopolysaccharide, LPS) be that studying the most thorough also is modal biological pyrogen, the difficult problem that must face of each big pharmaceutical factory especially simultaneously, even because when injection other trace of pg level LPS, also can cause the pyrogen reaction of serious patient.Therefore, sensitive intracellular toxin diagnostic techniques reliably is very necessary.
The amebocyte (amoebocytes) of sea king crab has superpower susceptibility to LPS, and when gram negative bacterium was invaded, it can pass through series of enzymatic reactions, and external invading bacteria is produced the cascade agglutination reaction, makes the microorganism inefficacy (1,2) of invasion.This process comprises the C factor (Factor C) identification of proenzyme state and is activated in conjunction with intracellular toxin, after becoming the state of organized enzyme, B factor activator with the downstream, the B factor of activation is converted into Thrombin coagulase with proclotting enzyme, Thrombin coagulase is converted into solidifying egg white with coagulagen, solidifying egg white is cross-linked with each other dehydration and forms gel (3,4).
Whether the tachypleus amebocyte lysate of utilizing extra large limulus blood amebocyte solute to make can be accurately and rapidly contains bacterial endotoxin in the qualitative or detection by quantitative sample.From the mid-1970s in last century, this intracellular toxin testing method begins for field of medicaments, and is extensively adopted by countries in the world very soon, and Chinese Pharmacopoeia and American-European pharmacopeia are decided to be legal bacterial endotoxins test with it.Through development and the improvement of decades, the tachypleus amebocyte lysate test method for endotoxin has been applied to each big field (5) such as pharmacy, medicine equipment, non-oral preparations, pharmaceutical products, biotechnological formulation, water quality, food inspection and scientific research.
Four kinds of extra large king crabs are only arranged at present in the world, i.e. U.S. king crab (Limulus polyphemus), Tachypleus tridentatus (Tachypleus tridentatus), tachypleus gigas (Tachypleus gigas) and rounded tail king crab (Carcinoscorpius rotundicauda).And can be used in that tachypleus amebocyte lysate produces two kinds of U.S. king crab and Tachypleus tridentatus are only arranged, corresponding tachypleus amebocyte lysate is referred to as LAL (Limulus amoebocyte lysate) and TAL (Tachypleus Amebocyte Lysate) respectively, the two reaction principle and effect are identical, but the difference that also has physicochemical property, as with (6) such as optimum pHs of intracellular toxin reaction.
Though tachypleus amebocyte lysate detection method (LAL/TAL) has highly sensitive (can detect fly gram level an intracellular toxin), advantage such as quick, because the cell lysate of employing complicated component as reaction raw materials, has the defective that can't overcome equally.Such as, non-specific interference problem: tachypleus amebocyte lysate also can be reacted (1) with (1-3)-callose except reacting with intracellular toxin.(1-3)-callose is a kind of polysaccharide that extensively exists in fungi, yeast, mushroom, higher plant, is the structure macromole that constitutes cell walls.Therefore be easy to run into the non-specific interference of (1-3)-callose in the tachypleus amebocyte lysate testing process, cause false positive results, this is for the medicine of complicated component, and especially Chinese medicine is a very big detection challenge (7).For another example, batch stability problem: tachypleus amebocyte lysate adopts extra large limulus blood as raw materials for production, because differences such as season, region make batch difference of tachypleus amebocyte lysate become common phenomenon.And the population quantity sharply minimizing in recent years owing to reasons such as environmental pollution, overfishing sea king crab approaches extinction (8), makes the starting material of production tachypleus amebocyte lysate face deficient day by day danger.
The main component of tachypleus amebocyte lysate is a series of serine stretch protein proenzyme (9,10), wherein the C factor is key molecule in the intracellular toxin detection method, in the king crab hemagglutination reaction system of endotaxin mediate, its specific recognition intracellular toxin, and first is activated by intracellular toxin, thereby starts the coagulation cascade reaction (11,12) in the whole limulus blood cell.Therefore, with the key molecule C factor of tachypleus amebocyte lysate as the basis, utilize its identification and in conjunction with behind the intracellular toxin, be activated as the organized enzyme form by pepsinogen, thereby characteristics that can the hydrolysis luminous substrate in conjunction with chemistry/fluorescence radiation quantitative technique, can develop with the C factor and detect quantitative technique (13) as the intracellular toxin of single component, compare with traditional LAL determination techniques, the recombinant C factor has lower background, higher sensitivity (14) to intracellular toxin under identical analysis condition.
Intracellular toxin detection technique composition based on the C factor is single, can use biotechnology to produce and strict quality, thereby has avoided batch unstable that caused by starting material, has protected these ancient " living fossil " species of extra large king crab simultaneously.Particularly importantly, this new technology has been eliminated traditional tachypleus amebocyte lysate to the nonspecific reaction of (1-3)-callose, can strengthen the specificity that intracellular toxin detects greatly, reduces " false positive " result's probability of occurrence.In addition, by with endotoxic specific combination, the recombinant C factor can also further be applied to endotoxin removals such as pharmaceutical prod, biological products.
To sum up, utilize the bionic method heterogenous expression king crab C factor, to be applied to the product development of intracellular toxin detection reagent, have important clinical meaning and huge commercial value.
Reference:
1.Iwanaga，S.(1993)Curr?Opin?Immunol?5，74-82
2.Muta，T.，and?Iwanaga，S.(1996)Curr?Opin?Immunol?8，41-47
3.Ding，J.L.，and?Ho，B.(2001)Trends?Biotechnol?19，277-281
4.Iwanaga，S.，and?Lee，B.L.(2005)J?Biochem?Mol?Biol?38，128-150
5.Novitsky，T.J.(1994)J.Endotoxin?Res?1，253-263
6. high international politics, Yan Jin. (1998) Chinese Pharmaceutical Journal 33 (3), 162
7. Huo Qi record, Shao Hongxia. (2003) CHINA JOURNAL OF CHINESE MATERIA MEDICA 28 (03), 199-201
8.Widener，J.W.，and?Barlow，R.B.(1999)Biol?Bull?197，300-302
9.Muta，T.，and?Iwanaga，S.(1996)Curr?Opin?Immunol?8，41-47
10.Iwanaga，S.(1993)Curr?Opin?Immunol?5，74-82
11.Nakamura，T.，Morita，T.，and?Iwanaga，S.(1986)Eur?J?Biochem?154，511-521
12.Iwanaga，S.，Miyata，T.，Tokunaga，F.，and?Muta，T.(1992)Thromb?Res?68，1-32
13.Ding，J.L.，and?Ho，B.(2010)Subcell?Biochem?53，187-208
14.Ding，J.L.，and?Ho，B.(2001)Trends?Biotechnol?19，277-281
Summary of the invention:
The object of the invention is to provide a kind of method with the recombinant expressed Tachypleus tridentatus C of the insect cell factor.
For achieving the above object, the technical solution used in the present invention is:
1) the recombinant baculovirus donor plasmid pFASTBacHTA-FC that contains Tachypleus tridentatus C factor gene transforms and to contain the genomic competent escherichia coli cell DH10Bac of baculovirus;
2) screening and evaluation contain the reorganization rod granule (FC-Bacmid) of the C factor;
3) the extracting reorganization bacmid dna of the C factor of having recombinated;
4) the recombinated reorganization bacmid dna transfection Sf 9 insect cell of the C factor;
5) detect the expression of the C factor in insect cell, determine optimum expression time and infection multiplicity (MOI).
The present invention has the following advantages:
1. the present invention is incorporated into Tachypleus tridentatus C factor gene in the baculovirus genome first, and preparation contains the reorganization rod granule of the Tachypleus tridentatus C factor.
2. the present invention lays a good foundation for obtaining a large amount of activated recombinant C factors first in the expressed in insect cells Tachypleus tridentatus C factor.
Description of drawings:
Below in conjunction with drawings and Examples this patent is further specified.
Fig. 1 is the recombinated extracting of rod granule of the Tachypleus tridentatus C factor and the result that bacterium colony PCR identifies.-for being template with the blue colonies, as primer, this is negative contrast with M13 Primer F and M13 Primer R; 1 be with white colony as template, with M13Primer F and FC Primer R as primer; 2 be with white colony as template, with M13 Primer R and FC PrimerF as primer; 3-6, with white colony as template, with M13 Primer F and M13 Primer R as primer; The reorganization rod granule sample of last twice for purifying.M is the nucleic acid molecular weight label, is respectively 5kb from top to bottom, 3kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp, 100bp.
Fig. 2 is the result that the rod granule PCR of the Tachypleus tridentatus C factor identifies that recombinated.+ for being template with the pFASTBACHTA-FC plasmid, this positive contrast; 1-4 is to be template with the reorganization rod granule (FC-Bacmid) of purifying, the band that obtains as primer PCR with FC Primer F and FC PrimerR; M is the nucleic acid molecular weight label, is respectively 5kb from top to bottom, 3kb, 2kb, 1.5kb, 1kb, 750bp and 500bp.
Fig. 3 has recombinated to detect the expression of the recombinant C factor in the cell behind the bacmid dna transfection Sf9 cell of the Tachypleus tridentatus C factor.Swimming lane 1 is the Sf9 cell of untransfected bacmid dna, this negative contrast; 2 for transfection the cell of positive control bacmid dna (recombinant protein molecular weight～90kDa); 3 for transfection the cell of 5 μ g reorganization bacmid dnas (FC-Bacmid); 4 for transfection the cell of 10 μ g reorganization bacmid dnas (FC-Bacmid).Behind the transfectional cell 72 hours, use the expression of the anti-His antibody test reorganization Tachypleus tridentatus C factor by the method for Western Blot.
Embodiment:
The recombinant plasmid pFASTBacHTA-FC that embodiment 1 contains the Tachypleus tridentatus C factor transforms the DH10Bac competent cell:
The pFASTBacHTA-FC plasmid of getting 2 μ l concentration and be 0.1 μ g/ μ l transforms 100 μ l intestinal bacteria DH10Bac competent cells, puts 30min on ice, and 42 ℃ of heat shock 90s are put 5min on ice rapidly, adds 1ml LB, is coated with three resistance LB flat boards behind 37 ℃ of recovery 3.5hr.37 ℃ of colors of cultivating observation bacterium colony behind the 40hr.
Wherein three resistance LB flat boards contain 50 μ g/ml Kanamycin, 7 μ g/ml Gentamicin and 10 μ g/mlTetracycline, and 100 μ g/ml Bluo-gal and 40 μ g/ml IPTG, flat board is put 4 ℃ and is kept in Dark Place.
Choose the white single bacterium colony described in example 1, draw the dull and stereotyped cultivation of LB again, draw continuously two to three times, till not producing blue colonies.Choose the bacterium colony that remains white after drawing continuously several times in the LB test tube that contains three resistances 37 ℃ cultivate 4 hours after, be that template is identified positive colony with the PCR method with bacterium liquid.
PCR system: F:100pmol, R:100pmol, dNTPs: each 50pmol, 5 * Prime star archaeal dna polymerase buffer:10 μ l, Prime star archaeal dna polymerase: 5U, sterilized water supply 50 μ l.
The PCR reaction conditions: with above-mentioned PCR reaction system mixing, fs 95 ℃ of pre-sex change 3min; 95 ℃ of subordinate phase, 1min; 54 ℃, 1min; 72 ℃, 3min20s; Carry out 30 circulations altogether; Phase III 72 ℃ of extensions 10min.
Primer is:
FC?Primer?F：5’-ATG?GTC?TTA?GCG?TCG?TTT?TT-3’
FC?Primer?R：5’-AAT?GAA?CTG?CCT?AAT?CCA?TG-3’
M13?Primer?F：5’-GTTTTCCCAGTCACGAC-3’
M13?Primer?R：5’-CAGGAAACAGCTATGAC-3’
Choose through PCR and be accredited as positive single bacterium colony to containing 50 μ g/ml Kanamycin, in the 5ml LB substratum test tube of 7 μ g/ml Gentamicin and 10 μ g/ml Tetracycline, 37 ℃ of shaking culture 24 hours; Get 1.5ml bacterium liquid, 12000rpm, the centrifugal 2min of room temperature removes supernatant; With 300 μ l solution I (15mM Tris-HCl, pH 8.0,10mMEDTA, 100 μ g/ml RNase A) resuspended thalline gently, add 300 μ l solution II (0.2N NaOH, 1% SDS) again, softly rotate mixing, room temperature is placed 5min; Slowly adding 300 μ l concentration is the potassium acetate (pH 5.5) of 3M, and the limit edged is mixing gently, puts 5-10min on ice then; The centrifugal 10min of 12000rpm carefully is transferred to 800 μ l supernatants in the EP pipe that has added 800 μ l Virahols, and mixing is put 5-10min on ice then gently; 12000rpm, the centrifugal 15min of room temperature; Supernatant discarded adds the ethanol of 500 μ l 70%, and upset is several times with washing and precipitating, 12000rpm, the centrifugal 15min of room temperature; Repeat once; Careful supernatant discarded allows be deposited in air drying 5-10min, uses 40 μ l TE damping fluid dissolution precipitations then.This is the bacmid dna of the C factor of having recombinated, and freezes in-20 ℃ standby.
The Sf9 cell presorts to six orifice plates (9 * 10
5Cells/well), 27 ℃ of pre-2hr that cultivate; Get Grace ' the s substratum mixing that 5 μ l or 10 μ l (1 μ g/ μ l) FC-bacmid and 100 μ l do not contain serum and additive respectively; Get Grace ' the s substratum mixing that 6 μ l transfection reagent Cellfectin and 100 μ l do not contain serum and additive; The plasmid that above-mentioned dilution is good and transfection reagent mixing, incubated at room 20min; While washes six orifice plate cells and discards washing lotion with Grace ' the s substratum that 2ml does not have added ingredients.Grace ' the s substratum of getting 0.8ml and do not have added ingredients adds in the six orifice plate cells after the washing, adds the mixture of plasmid and transfection reagent again, shakes up gently.Establish a positive control and negative control simultaneously.Change after the transfection 5 as a child and contain serum and antibiotic substratum, cultivated 72 hours for 27 ℃, low-speed centrifugal is collected culture supernatant, has namely obtained the P1 viral sample of the reorganization Tachypleus tridentatus C factor.
The viral liquid of the P1 of gained is diluted 2-3 gradient successively to be added on the Sf9 monolayer cell that discards substratum, effect 1hr, supernatant is removed in suction, to contain the perfect medium of serum and 2.2% low melting-point agarose by 1: 1 mixed, add X-gal to many concentration be 150 μ g/ml, be added on the cell fast immediately, after gelling admittedly, be inverted 6 orifice plates, place 27 ℃ of incubators to cultivate, occur up to plaque.Calculate the plaque number, by formula titre (pfu/ml)=(1/ extent of dilution) * plaque number, determine virus titer with this.
With P1 virus infection 2 * 10
6Be in the Sf9 cell of logarithmic phase, place 27 ℃ of incubators to cultivate, up to the complete cracking of cell, low-speed centrifugal is removed cell debris, preparation infectious titer liquid; With 10 times gradients infectious titer liquid is diluted to 10
-6With 10
-7, do the plaque analysis respectively, calculate the plaque number, by formula titre (pfu/ml)=(1/ extent of dilution) * plaque number, determine virus titer with this;
Inoculate volume (ml)=MOI (the pfu/cell) * cell count ÷ titre (pfu/ml) of required infectious titer liquid again according to formula, calculating MOI is 5,7,10 o'clock, needs the volume of inoculation infectious titer liquid; Establish the not negative contrast of the Sf9 cell of virus inoculation liquid simultaneously; After infecting 72hr, receive cell, the sample of preparation polyacrylamide gel electrophoresis.
The method of embodiment 6 usefulness Western Blot detects the expression of the Tachypleus tridentatus C factor in insect cell:
When the Sf9 insect cell is in logarithmic phase, get reorganization rod granule (FC-Bacmid) cells infected that contains Tachypleus tridentatus C factor gene of an amount of high titre, behind 27 ℃ of cultivation 72hr, cell is blown afloat, 500g low-speed centrifugal 5min abandons supernatant, and it is resuspended that cell precipitation adds 1ml PBS, get 300 μ l, the centrifugal 5min of 500g, the exhaustion supernatant adds 100 μ l, 1 * SDSloading buffer, the vortex mixing is to clarification, 100 ℃ are boiled 10min, centrifugal, get sample on the 10 μ l supernatants, carry out SDS-PAGE, gel density is 12%.Change film, constant voltage 100V, 2 hours with wet shifting method behind the electrophoresis.Film is taken out from electric turn trough, and deionized water is rinsing a little, is immersed in the 5% skimmed milk confining liquid room temperature and slowly sways one hour.Add anti-His primary antibodie diluent, jog was hatched 1 hour under the room temperature.After primary antibodie is hatched end, use PBST damping fluid rinsing film three times, each 10 minutes.Add two then and resist room temperature jog one hour.Two anti-hatch end after, with embathing again three times behind the PBST rinsing film.Detect with horseradish peroxidase HRP-ECL luminescence method then.Wet commentaries on classics makes electricity consumption change the liquid collocation method: Tris-base 3.0g, and glycine 14.4g, methyl alcohol 200mL adds deionized water to 1000mL.PBST：1×PBS，Tween-200.01％。Confining liquid and antibody diluent are the PBST that contains 5% skim-milk.
Claims (4)
1. the expression method of the Tachypleus tridentatus C factor in insect cell is characterized in that:
1) the recombinant baculovirus donor plasmid pFASTBacHTA-FC that contains Tachypleus tridentatus C factor gene transforms and to contain the genomic competent escherichia coli cell DH10Bac of baculovirus;
2) contain screening and the evaluation of the reorganization rod granule (FC-Bacmid) of C factor gene;
3) contain the extracting of the reorganization bacmid dna of C factor gene;
4) contain the reorganization rod granule transfection insect cell of C factor gene;
5) detect the expression of the C factor in insect cell, determine optimum expression time and infection multiplicity.
2. by the expression method of the described Tachypleus tridentatus C of claim 1 factor in insect cell, it is characterized in that:
A) the described recombinant expression plasmid pFASTBacHTA-FC that contains the C factor gene transforms the method for DH10Bac, it is characterized in that: the pFASTBacHTA-FC plasmid of getting 2 μ l concentration and be 0.1 μ g/ μ l transforms 100 μ l intestinal bacteria DH10Bac competent cells, put 30min on ice, 42 ℃ of heat shock 90s, put 5min on ice rapidly, add 1ml LB, be coated with three resistance LB flat boards (50 μ g/ml Kanamycin behind 37 ℃ of recovery 3.5hr, 7 μ g/ml Gentamicin and 10 μ g/ml Tetracycline), 37 ℃ of colors of cultivating observation bacterium colony behind the 40hr;
B) described screening and the authentication method that contains the reorganization rod granule of C factor gene, it is characterized in that: choose as the white single bacterium colony described in a), again draw dull and stereotyped the cultivation, draw two to three times continuously, till not producing blue colonies, choose the bacterium colony that remains white after drawing continuously several times in the LB test tube that contains three resistances 37 ℃ cultivate 4 hours after, identify positive colony with the PCR method;
PCR system: F:100pmol, R:100pmol, dNTPs: each 50pmol, 5 * Prime star archaeal dna polymerase buffer:10 μ l, Prime star archaeal dna polymerase: 5U, sterilized water supply 50 μ l;
The PCR reaction conditions: with above-mentioned PCR reaction system mixing, fs 95 ℃ of pre-sex change 3min; 95 ℃ of subordinate phase, 1min; 54 ℃, 1min; 72 ℃, 3min20s; Carry out 30 circulations altogether; Phase III 72 ℃ of extensions 10min;
Primer is:
FC?Primer?F：5’-ATG?GTC?TTA?GCG?TCG?TTT?TT-3’
FC?Primer?R：5’-AAT?GAA?CTG?CCT?AAT?CCA?TG-3’
M13?Primer?F：5’-GTTTTCCCAGTCACGAC-3’
M13?Primer?R：5’-CAGGAAACAGCTATGAC-3’
C) the described extracting that contains the reorganization bacmid dna of C factor gene, it is characterized in that: choose through PCR and be accredited as positive single bacterium colony to containing 50 μ g/ml Kanamycin, in the test tube of the 5ml LB substratum of 7 μ g/ml Gentamicin and 10 μ g/ml Tetracycline, cultivated 24 hours for 37 ℃; Get the centrifugal supernatant that goes of 1.5ml bacterium liquid, with 300 μ l solution I (15mM Tris-HCl, pH 8.0,10mM EDTA, 100 μ g/ml RNase A) resuspended thalline gently adds 300 μ l solution II (0.2N NaOH again, 1%SDS), softly rotate mixing, room temperature is placed 5min; Slowly adding 300 μ l concentration is the potassium acetate (pH 5.5) of 3M, and the limit edged is mixing gently, puts 10min on ice then; The centrifugal 10min of 12000rpm carefully is transferred to 800 μ l supernatants in the EP pipe that has added 800 μ l Virahols, and mixing is put 10min on ice then gently; 12000rpm, the centrifugal 15min of room temperature; Supernatant discarded adds the ethanol of 500 μ l 70%, and upset is several times with washing and precipitating, 12000rpm, the centrifugal 15min of room temperature; Repeat once; Careful supernatant discarded allows be deposited in air drying 10min, uses 40 μ l TE damping fluid dissolution precipitations then.This is the bacmid dna of the C factor of having recombinated, and freezes in-20 ℃ standby.
3. by the expression method of the described Tachypleus tridentatus C of claim 1 factor in insect cell, it is characterized in that: contain the reorganization bacmid dna transfection Sf 9 insect cell of Tachypleus tridentatus C factor gene and the method that virus titer is determined in the plaque analysis, its working method is: the Sf9 cell presorts to six orifice plates (9 * 10
5Cells/well), 27 ℃ of pre-2hr that cultivate; Get Grace ' the s substratum mixing that 5 μ l or 10 μ l (1 μ g/ μ l) FC-bacmid and 100 μ l do not contain serum and additive respectively; Get Grace ' the s substratum mixing that 6ul transfection reagent Cellfectin and 100 μ l do not contain serum and additive; The plasmid that above-mentioned dilution is good and transfection reagent mixing, incubated at room 20min; While washes six orifice plate cells and discards washing lotion with Grace ' the s substratum that 2ml does not have added ingredients; Grace ' the s substratum of getting 0.8ml and do not have added ingredients adds in the six orifice plate cells after the washing, adds the mixture of plasmid and transfection reagent again, shakes up gently.Establish a positive control and negative control simultaneously; Transfection is changed after 5 hours and is contained serum and antibiotic substratum, cultivates 72 hours for 27 ℃, and low-speed centrifugal is collected culture supernatant, has namely obtained the P1 viral sample of the reorganization Tachypleus tridentatus C factor;
The viral liquid of the P1 of gained is diluted 2-3 gradient successively to be added on the Sf9 monolayer cell that discards substratum, effect 1hr, supernatant is removed in suction, to contain the perfect medium of serum and 2.2% low melting-point agarose by 1: 1 mixed, add X-gal to many concentration be 150 μ g/ml, be added on the cell fast immediately, after gelling admittedly, be inverted 6 orifice plates, place 27 ℃ of incubators to cultivate, occur up to plaque; Calculate the plaque number, by formula titre (pfu/ml)=(1/ extent of dilution) * plaque number, determine virus titer with this.
4. by the expression method of the described Tachypleus tridentatus C of claim 1 factor in insect cell, it is characterized in that: determine the optimum expression time of the C factor and the working method of infection multiplicity (MOI):
A) with P1 virus infection 2 * 10
6Be in the Sf9 cell of logarithmic phase, place 27 ℃ of incubators to cultivate, up to the complete cracking of cell, low-speed centrifugal is removed cell debris, preparation infectious titer liquid; With 10 times gradients infectious titer liquid is diluted to 10
-6With 10
-7, do the plaque analysis respectively, calculate the plaque number, by formula titre (pfu/ml)=(1/ extent of dilution) * plaque number, determine virus titer with this;
Inoculate volume (ml)=MOI (the pfu/cell) * cell count ÷ titre (pfu/ml) of required infectious titer liquid again according to formula, calculating MOI is 5,7,10 o'clock, needs the volume of inoculation infectious titer liquid; Establish the not negative contrast of the Sf9 cell of virus inoculation liquid simultaneously; After infecting 72hr, receive cell, the protein sample of preparation polyacrylamide gel electrophoresis.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119271A (en) * | 2016-07-04 | 2016-11-16 | 长春理工大学 | A kind of DNA recombinant expression Tachypleus tridentatus C factor specific detection endotoxin strengthens turbidimetry |
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| US6645724B1 (en) * | 1997-09-19 | 2003-11-11 | National University Of Singapore | Assays for endotoxin |
| CN1529711A (en) * | 2001-06-28 | 2004-09-15 | ά | Methods and reagents for detecting endotoxin |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6645724B1 (en) * | 1997-09-19 | 2003-11-11 | National University Of Singapore | Assays for endotoxin |
| CN1529711A (en) * | 2001-06-28 | 2004-09-15 | ά | Methods and reagents for detecting endotoxin |
Non-Patent Citations (2)
| Title |
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| 孙向军 等: "东方鲎FactorC中的结构域在结合脂多糖中的作用", 《生物化学与生物物理进展》 * |
| 魏京广 等: "鲎C 因子的研究进展", 《安徽农业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119271A (en) * | 2016-07-04 | 2016-11-16 | 长春理工大学 | A kind of DNA recombinant expression Tachypleus tridentatus C factor specific detection endotoxin strengthens turbidimetry |
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